Electron microscopy tissue grid staining and storing rack and method

ABSTRACT

An apparatus and method for staining, washing and storing a plurality of grid mounted electron microscopy tissue specimens are based upon placing the grids having the mounted tissue in a rack which isolates and provides an identifying file indicia and fluid passages for staining and washing each of a plurality of grids. The same rack also receives a protective cover for grid storage thereby eliminating much individual grid handling and the requirement for separate staining and storage apparatus.

United States Patent 1 Kinney et a1.

ELECTRON MICROSCOPY TISSUE GRID STAINING AND STORING RACK AND METHOD Inventors: Thomas D. Kinney, 3120 Devon Rd.; John E. P. Pickett, 3323 Pinafore Dr., both of Durham, NC.

Filed: Sept. 18, 1972 Appl. No.: 290,170

U.S. Cl. 356/244, 350/95 Int. Cl. G0ln 21/16 Field of Search 356/244, 246, 36,

References Cited UNITED STATES PATENTS Marton 250/495 B Oct. 30, 1973 3/1971 Metzgar et a1 356/244 3,572,892 3,649,464 3/1972 Freeman 350/95 3,656,833 4/1972 Wallace 356/244 Primary Examiner-William L. Sikes Attorney-B. B. Olive [57] ABSTRACT An apparatus and method for staining, washing and storing a plurality of grid mounted electron microscopy tissue specimens are based upon placing the grids having the mounted tissue in a rack which isolates and provides an identifying file indicia and fluid passages for staining and washing each of a plurality of grids. The same rack also receives a protective cover for grid storage thereby eliminating much individual grid handling and the requirement for separate staining and storage apparatus.

11 Claims, 22 Drawing Figures Patented Oct. SU, 1973 3,768,914

5 Shoots-Shoot I 7' IIIIIIIIIIIIIII.

Patented =Oct. 30, 1973 v 3,768,914

3 Sheets-Sheet 2 Patented Oct. 30, 1973 3,768,914

3 Sheets-Sheet f5 ///H//r//A/- FIG. 21 4 1 ELECTRON MICROSCOPY TISSUE GRID STAINING AND STORING RACK AND METHOD BACKGROUND OF THE INVENTION copy it has been difficult to develop staining procedures that are specific for particular substances, but a general stain to increase contrast is very helpful, particularly when the contrast is low. Most staining is done with reagents containing metal ions to increase the electron density of the tissue structures. Stains include lead citrate, uranyl acetate, phosphotungstic acid, phosphomolybdic and ammonium molydate.

It has been the practice to mount electron microscopy tissue specimens on small diameter, e.g., approximately three thirty-seconds inch, copper grids, e.g., 200 mesh. After the mounting procedure, each minute grid having a tissue specimen is placed with a fine pair of forceps on a drop of stain and is allowed to stay for a predetermined period, e.g., 5 minutes. Each grid is then carried with the forceps through several distilled water rinses. Through all of these individual operations on each grid, provision must be made for maintaining the identity of each grid. It is also often desirable that particular groups of grids receive as near as possible the same staining exposure to standardized results.

It would be desirable to stain electron microscopy sections on grids in volume in order to speed up processing and also give better and more uniform quality material for electron microscopy study. However, prior art practices have over a long period of time followed the individual grid staining and washing procedures described above.

In addition to the mounting, storing, and washing procedures mentioned above in which grid identification, freedom from contamination and physical protection are most important for the very delicate grids, it also becomes important that the grids be suitably protected and identified when stored after examination so that they may be retrieved if necessary for other examinations. i

There is now sold under the trademark LKB what is called a Specimen Grid Box for use in storing, by a file number, electron microscopy grids having tissues mounted thereon. The manufacturer is LKB-Produkter A B, Stockholm-Bromma 1, Sweden. This grid box comprises a holder made of plastic having a transparent sliding cover. The cover includes a pair of integral, inturned flanges and receives a mating flanged grid storage container which has twenty rows of diamond shaped holes or wells which are arranged in ten columns. The rows each have five wells and the rows and columns are marked lengthwise with letters and laterally with numbers so that each well has an identification such as A-S." This allows the technician to record where a particulargrid is stored. Each LKB grid box also has a serial number on one end of the container such as the number Box 52134" which allows the box to be identified. The diamond shaped well is specifically designed, dimension and shapewise, so as to receive the grid periphery by opposed well edges and to allow the grid to be stored without the delicately mounted .tissue touching the well surfaces.

It is. significant to note that the LKB type grid box container is designed to that the grid storage wells are open at one end but are closed at'the opposite end. it should also be noted that the sliding cover is designed with a relatively close sliding fit over the open'end'of the wells. Thus, it can be appreciated that while the conventional LKB type grid box does serve as a useful storage and filing device for electron microscopy grids, it has no utility forstaining or washing procedures.

From the foregoing, it can be seen that the prior art methods and apparatus for the very delicate handling of electron microscopy grids during storage, staining and washing procedures have required individual grid handling in all steps and have required separate apparatus for storage as distinct from staining and washing. Thus, the prior art would benefit from an apparatus which is adapted to serve both the'purpose of being a file storage rack as well as a staining and washing rack and from a method which substantially reduces the amount and hazards of individual grid handling.

SUMMARY OF THE INVENTION The invention is directed to a method and apparatus based upon utilization of a container adapted to provide a plurality of indicia identified wells or holes for staining and washing of electron microscopy grids and to also provide means whereby the'grids so stained may be stored. In one embodiment, the grids are stored in a container having a plurality of wells adapted on one side with open ends to receive and provide identification for an equal number of electron microscopy grids and on the other side to provide small holes communicating with the wells for transfer of staining and washing fluids. During staining, a cover having small holes corresponding with the wells covers the grid receiving side of the container. This container once so covered is fitted vertically into a receptacle which receives a selected staining fluid so that the grids may be stained and then washed in another similar receptacle having a washing fluid. For storage, a pair of'imperforate covers are slidably received by the container on both sides of the wells which secures and covers all the grids for indefinite storage. 7

In a second embodiment, the container which is used in the first embodiment is fitted with a porous pad which provides a porous closure for one end of the wells and is fitted with an imperforate cover on the other, open end, side of the wells for staining and washing procedures. For storage purposes, the porous pad is removed and replaced by an imperforate cover so that both ends of the wells are covered by imperforate covers during storage.

A third embodiment utilizes a modification of the LKB grid storage box in which the conventional grid box container is provided on one side with a porous pad which provides porous ends for the wells so that when the grid box is mounted in its conventional storage cover and the grid box is placed in a suitable container of staining or washing fluid, a fluid procedure may be effected. Another cover is provided to cover the wells during storage.

A fourth embodiment is based upon providing the normally closed end of each of the wells in a LKB type grid storage box with an aperture which, with the conthat the grids remain in the same base container during staining, washing, and storage. It is further important to recognize that unlike the prior art approach of individually fluid treating each grid, the grids are processed in volume.

DESCRIPTION OF THE DRAWINGS FIG. 1 is an exploded view of the first embodiment base grid container and perforated cover components used during staining and washing.

FIG. 2 is a perspective view of a fluid container used to hold rinse and wash solutions and which mates with the base container.

FIG. 3 is a perspective view of the first embodiment components as they appear assembled for staining and washing. a

FIG. 4 is a top view of the FIG. 2 container with the FIG. 3 assembly installed.

FIG. 5 is a longitudinal section view taken in the direction 5-5 of FIG. 3.

FIG. 6 is a cross section view taken in the direction 6-6 of FIG. 3.

FIG. 7 is an enlarged cross sectional portion of FIG. 5 showing the typical disposition of several grids.

FIG. 8 is an exploded view of the first embodiment components used for storage.

FIG. 9 is a cross section view taken through the assembled components of FIG. 8.

FIG. 10 is an exploded view of the components used in the second embodiment for staining and washing as well as storage.

FIG. 11 is a perspective view of the second embodiment components assembled for staining and washing procedures.

FIG. 12 is a side view of the FIG. 11 assembly installed in a fluid container of the FIG. 2 type.

FIG. 13 is a top view of the assembly of FIG. .12.

FIG. 14 is a cross section view taken in the direction 14-14 of FIG. 11.

FIG. 15 is a longitudinal section view taken in the direction 15-15 of FIG. 11.

FIG. 16 is an enlarged cross sectional portion of FIG. 15 showing the typical disposition of several grids.

FIG. 17 is an exploded view of the components used in the third embodiment for staining and washing as well as storage.

FIG. 18 is a longitudinal section view of the third embodiment components assembled. for staining and' DESCRIPTION OF THE PREFERRED EMBODIMENT The preferred embodiment is shown in FIGS. 1

through 9, a second embodiment in FIGS. 10 through 16, a third embodiment IN FIGS. 17 through 20, and a fourth embodiment in FIGS. 21 and 22. Since the third and fourth. embodiments more closely follow prior artpractices these will be first described and then a description will be given for comparison of the second and finally of the first and preferred embodiment.

Referring further to FIGS. 17 through 20, the third embodiment represents a simple but significant modification of the LKB type grid storage box. The conventional LKB type storage box includes a solid cover 30 which normally does not contain the holes 31 shown in FIG. 17. These holes are of the smallest diameter which allows fluid flow. Cover 30 normally slides over a container 32 having'the diamond shaped wells 33 and the vertical and horizontal indicia, l, 2, 3, 4, 5, A, B, C, etc., shown in FIG. 17. Container 32 does not normally have the slot 41 which receives the porous pad 40. Thus, cover 30 and container 32 represent the conventional LKB grid storage box except for the presence of the holes 31 in cover 30 and except for the presence of the slot 41 and porous pad 40 in container 32. Also, the supplementary storage cover 34, shown in FIG. 17, is not provided with the conventional LKB storage box since cover 30, without holes 31, serves the same purpose during storage when installed on container 32 in its conventional form.

Slot 41 is cut out of the normally solid side of container 32. As best seen in FIGS. 18 through 20, the for mation of slot 41 to receive pad40 causes what appear in those figures as the bottom ends of the grid receiving walls to be opened and covered by pad 40. A variety of porous materials may be used for pad 40. A preferred material for pad 40 which provides both filtering and fluid passage is identified as Labpor porous polyethelene plastic sheets one-sixteenths inch thick, micron porosity, hydrophically treated, made by Porex Company of Fairborn, Georgia. Pad 40 is suitably rigidly secured to container 32. When using this third embodiment, the grids having mounted tissue are placed in their respective wells 33, cover 30 having holes 31 is installed and'the assembled grid box is placed in a stain solution container such as indicated in FIG. 2 and which contains the appropriate stain. Holes 31 and pad 40 provide fluid passages. Here it should be mentioned that successful staining and rinsing has been achieved without holes 31 in cover 30 and with only the presence of pad 40 as a means of fluid passage. This alternate thirdembodiment does appear, however, to be more time consuming with such experience as has been gained to date. Under normalprocedures, after staining, each grid is handled individually with forceps and is passed through several rinses of distilled water. Such procedures subject the very delicate and small, e.g., three-thirty-seconds inch, grids to damage and contamination at every step. With the present invetion, however, the delicate and small grids remain in container 32 and may be washed through several separate rinses and dried while remaining in the grid box, thus eliminating much of the hazard previously experienced.

Once the particular sections on the grids have been stained, rinsed, and individually examined under an electron microscope as required, the grids may then be returned to the same grid box and solid cover 34 installed which now becomes a permanent storage by box and file designation. Thus, each grid, i'.e., each tissue section, may be assigned a box number, e.g., F-l235 shown in FIG. 17 and a file number in the box, e.g., A-3, which it may retain for future reference for the original tissue mounting, the staining, examination and subsequent storage. Thus, box F-1235 may be referenced with regard to a staining technique whereas before each individual grid had to be so referenced and the staining techniques could not be uniform for a group of related grids.

From what has been described, it can be seen that the presence of fluid passages into the wells in container 32 allows the conventional LKB type grid storage box to be used for both staining and washing of large numbers of grids and with solid cover 34 installed allows the grids to be safely stored in the same container 32 in which they were stained. This simple modification in both structure and procedure thus produces significant advantages.

The fourth embodiment shown in FIGS. 21 and 22,

, like the third embodiment, basically constitutes a modification of the LKB type grid storage box. In particular, the modified LKB grid box container 32' has holes 50 through the normally closed base ends of each grid receiving well 33' in the container 32'. Thus, container 32'may be identical to the container portion of the conventional LKB grid box except for the fact that the normally closed ends of the grid receiving wells 33 are apertured with holes 50. The diameter of holes 50 should be of the minimum size necessary to provide fluid flow. Additionally, for purposes of the fourth embodiment, it is desirable to provide shallow longitudinal slots 51 on the underside of the cover 30' and which are aligned lengthwise with the rows of grid receiving wells 33 to provide additional fluid passages. Slots 51 may, for example, be about one thirty-second inch wide and one thirty-second inch deep.

In using the fourth embodiment, the delicate and small size grids with the tissue mounted thereon are individually installed as previously explained in the respective apertured wells 33' in the container 32. The slotted cover 30' is installed and then the assembled grid box is placed vertically in a fluid container such as shown in FIG. 2 and having the desired staining solution. During staining, the staining fluid reaches and stains the tissue sections by flowing through the holes 50 and the slots 51. After the grids have been in the staining solution for the required time they are retained in the grid box and passed through other containers containing distilled water as required to complete washing. While still remaining in the grid box, the grids are allowed to dry and are then available for examination and eventual return to the grid box for storage. A cover such as cover 34 in FIG. 17 is installed and adapts the grid box for permanent storage under a suitable box number and file indicia as previously explained.

With the above description in mind, reference is next made to the second embodiment shown in FIGS. through 16. The second embodiment, like the third and fourth embodiments is directed to providing a grid box which can serve both as a staining and washing rack as well as a storage rack. Additionally, the grid box of the second embodiment is adapted to be mated with a container such as shown in FIG. 2 which serves in place of a beaker to hold stain and wash solutions and positions pletely through thecentral plate portion 61. Diamond shaped wells such as used in the LKB box are preferred, as previously mentioned, since they tend to support the respective grids on thegrid peripheries and prevent the mounted tissue from touching the sides ofthe wells. Wells of other shapes may, of course be used. Integral with plate portion 61 grid box includes on the well grid receiving side a pair of slotted flanges 62, 63 which slidably receive an imperforate cover 65 and engage stop protrusions 66, 67. The fit between cover 65 and the face of plate portion 61 should preferably allow at least some fluid passage. In one embodiment, a

polyethelene plastic sheet I-% inch wide by 2 inches long by one-sixteenth inch thick has been employed for cover 65. On an opposite side slotted flanges 70, 71 slidably receive a replaceable and cleanable porous pad 72 which provides a bottom surface. A Labpor onetenth inch thick sheet, 120 micron porosity, hydrophically treated sheet as made by Porex Company, Fairborn, Georgia, of a length and width similar to that of cover 65 has been employedsuccessfully as pad 72. Unlike pad 40 in FIG. 17 which is intended to be permanently secured, pad 72 is'removably secured and, thus, can be cleaned.

With pad 72 in place, the grids positioned and identified by appropriate indicia 75, e.g., numbers l-25, and cover 65 installed, the grid box as shown in FIGS. 12-13 is next placed in a mating open top container (like the container of FIG. 2) having sidewalls 81, 82, end walls 83, 84 and bottom wall 85. End walls 83, 84 are provided with slots 86, 87 adapted to slidably receive the flange portions 88, 89 (FIG. 11) ofgrid box 60 and during staining and washing to maintain the grid box in a vertical position. A tab portion 90 is formed integral with box 60 and provides a convenient means for installing and removing the grid box assembly.

After the grids have been appropriately stained, washed and dried while remaining in grid box 60 and after the required individual examination under an electron microscope, pad 72 is removed, solid cover 65 is installed and a similar solid cover 91 (FIG. 10) is substituted for pad 72. In this configuration, the grid box assembly becomes a storage assembly but at no time must any one of the individual and very delicate grids be removed from box 60 except when being examined. A suitable file box number, e.g., Z-l234, may be used as shown in FIGS. 10 and 11.

The first and preferred embodiment is shown in FIGS. 1 through 9 and its construction and use will be immediately understood from what has already been explained. The grid box assembly used for staining and washing comprises the grid box 100, the perforated cover 101 and the fluid container 102. Grid box is generally similar to the previouslyexplained grid box 60 except that a porous pad, like pad 40 or pad 72, is not employed and the individual wells 103 instead of being of a uniform diamond shape size throughout the thickness of the grid box, as in FIG. 16, have closed ends which in turn are provided with small holes 105 like holes 50 (FIG. 22) as best shown in FIG. 7. Thus, with the grids installed as in FIG. 7 and with the perforated cover 101 installed, the cover holes 106 and the well holes 105 insure a fluid exchange path for both staining and washing fluids when installed in the container of FIG. 2 and as depicted in FIG. 4.

Once the grids have been stained and washed as a group and then individually examined as required, storage is effected by installing a pair of solid, relatively snug fitting, covers 110, 111 as shown in FIGS. 8 and 9. With such covers installed, the grid box stainingwashing assembly becomes a storage assembly for essentially indefinate storage. A suitable file box number, e.g., .M-462l, as shown in FIGS. 1, 3 and 8, may be used.

In all embodiments, it can be seen that individual handling of the delicate and small size grids during staining and washing has been eliminated. Thus, this extremely crucial, tedious and time consuming procedure has been abolished. Each grid box embodiment, furthermore, mates and is preferably used with a fluid receptacle that holds the box vertically in a uniform position during staining and washing and with minimum opportunity for damage to the grids. More uniform results are now assured with substantially less risk to the sometimes critically important minute tissue sections.

What is claimed is:

1. An assembly of cooperative laboratory components adapted to facilitate staining, washing and storage of grid mounted electron microscopy tissue specimens comprising, in combination:

a. a generally rectangular base container having a central plate portion providing a plurality of individual wells each adapted to receive in one open end thereof an electron microscopy specimen grid and to hold the grid by its peripheral edge whereby the specimen itself remains out of contact with well surfaces;

b. a first cover adapted to be removably secured to said base container, said base container and first cover being constructed for operational cooperation such that when said first cover is so secured said grids are retained in their respective wells by said first cover and each well is provided with at least one fluid exchange path having external communication for exchanging staining and washing fluids;

0. a fluid container adapted to hold a selected fluid and to slidably receive and maintain said base container and first cover in a vertical position during exchanges of fluids with said grids; and

d. a second cover means adapted to be secured to said base container in place of said first cover and being adapted in cooperation with said base container to enclose each said well to provide an-enclosed storage void for each said grid.

2. An assembly as claimed in claim 1 wherein said base container includes on said plate portion opposite the side thereof having the grid receiving ends of said wells an attached porous pad, said pad serving to provide a porous closed end for each said well and thereby establishing said fluid exchange path.

3. An assembly as claimed in claim 2 wherein said pad is fixedly secured to said plate portion.

4. An assembly as claimed in claim 2 wherein said pad is slidably secured to said base container adjacent i said plate portion.

5. An assembly as claimedin claim 3 wherein said first cover is provided with a plurality of holes of substantially smaller size than and mating with respective said wells to facilitate passage of fluids therethrough.

6. An assembly as claimed in claim 5 wherein said second cover means has integral portions adapted during storage to cover both said pad and the grid receiving side of said base container.

7. An assembly as claimed in claim 4 wherein said first cover is imperforate and including a further imperforate cover, said pair of imperforate covers being adapted to be slidably secured to said base container on opposite sides of said plate portion during storage of said grids.

8. An assembly as claimed in claim 1 wherein said first cover comprises a perforate cover having holes mating with said wells, wherein each said well on the end opposite the grid receiving end thereof is closed and provided with a fluid passage adapted to communicate with fluid sources externally thereof, and wherein said second cover means comprises a pair of imperforate covers adapted to be slidably secured to said base container during storage of said grids.

9. An assembly as claimed in claim 1 wherein said first cover is imperforate and said base container wells are closed at the ends opposite said open grid receiving ends and in such closed ends are provided with holes which provide for each respective well a said fluid exchange path.

10. An assembly as claimed in claim 1 wherein said first cover is provided with a plurality of holes of substantially smaller size than and mating'with respective said wells to facilitate passage of fluids therethrough and wherein said base container wells are closed at the ends opposite said open grid receiving ends and in such closed ends are provided with holes which provide for each respective well a said fluid exchange path.

11. The method of staining, washing and storing electron microscopy grids having tissue specimens mounted thereon, comprising the steps:

a. mounting a plurality of said grids' in a base container having a plurality of open ended grid receiving wells shaped to engage the grid peripheries and hold the tissue specimens out of contact with the well surfaces;

b. securing the grids in place by installing a cover on said base container over the wells open ends;

0. treating the plurality of said grids simultaneously with appropriate staining and washing fluids in sequence while confined in said base container;

d. drying and selectively examining said grids as required; and

e. covering said wells and the grids contained therein for storage by detachably securing imperforate covering means to said base container.

l i i t I 

1. An assembly of cooperative laboratory components adapted to facilitate staining, washing and storage of grid mounted electron microscopy tissue specimens comprising, in combination: a. a generally rectangular base container having a central plate portion providing a plurality of individual wells each adapted to receive in one open end thereof an electron microscopy specimen grid and to hold the grid by its peripheral edge whereby the specimen itself remains out of contact with well surfaces; b. a first cover adapted to be removably secured to said base container, said base container and first cover being constructed for operational cooperation such that when said first cover is so secured said grids are retained in their respective wells by said first cover and each well is provided with at least one fluid exchange path having external communication for exchanging staining and washing fluids; c. a fluid container adapted to hold a selected fluid and to slidably receive and maintain said base container and first cover in a vertical position during exchanges of fluids with said grids; and d. a second cover means adapted to be secured to said base container in place of said first cover and being adapted in cooperation with said base container to enclose each said well to provide an enclosed storage void for each said grid.
 2. An assembly as claimed in claim 1 wherein said base container includes on said plate portion opposite the side thereof having the grid receiving ends of said wells an attached porous pad, said pad serving to provide a porous closed end for each said well and thereby establishing said fluid exchange path.
 3. An assembly as claimed in claim 2 wherein said pad is fixedly secured to said plate portion.
 4. An assembly as claimed in claim 2 wherein said pad is slidably secured to said base container adjacent said plate portion.
 5. An assembly as claimed in claim 3 wherein said first cover is provided with a plurality of holes of substantially smaller size than and mating with respective said wells to facilitate passage of fluids therethrough.
 6. An assembly as claimed in claim 5 wherein said second cover means has integral portions adapted during storage to cover both said pad and the grid receiving side of said base container.
 7. An assembly as claimed in claim 4 wherein said first cover is imperforate and including a further imperforate cover, said pair of imperforate covers being adapted to be slidably secured to said base container on opposite sides of said plate portion during storage of said grids.
 8. An assembly as claimed in claim 1 wherein said first cover comprises a perforate cover having holes mating with said wells, wherein each said well on the end opposite the grid receiving end thereof is closed and provided with a fluid passage adapted to communicate with fluid sources externally thereof, and wherein said second cover means comprises a pair of imperforate covers adapted to be slidably secured to said base container during storage of said grids.
 9. An assembly as claimed in claim 1 wherein said first cover is imperforate and said base container wells are closed at the ends opposite said open grid receiving ends and in such closed ends are provided with holes which provide for each respective well a said fluid exchange path.
 10. An assembly as claimed in claim 1 wherein said first cover is provided with a plurality of holes of substantially smaller size than and mating with respective said wells to facilitate passage of fluids therethrough and wherein said base container wells are closed at the ends opposite said open grid receiving ends and in such closed ends are provided with holes which provide for each respective well a said fluid exchange path.
 11. The method of staining, washing and storing electron microscopy grids having tissue specimens mounted thereon, comprising the steps: a. mounting a plurality of said grids in a base container having a plurality of open ended grid receiving wells shaped to engage the grid peripheries and hold the tissue specimens out of contact with the well surfaces; b. securing the grids in place by installing a cover on said base container over the wells open ends; c. treating the plurality of said grids simultaneously with appropriate staining and washing fluids in sequence while confined in said base container; d. drying and selectively examining said grids as required; and e. covering said wells and the grids contained therein for storage by detachably securing imperforate covering means to said base container. 